The inability to obtain atomic structures on actin cytoskeleton has severely hindered our understanding of this most abundant eukaryotic protein and its dynamic turnovers in performing a myriad of cellular functions such as cell motility, cytokinesis and morphogenesis through interactions with hundreds of actin-binding proteins. This application describes an innovative double-mutant strategy to overcome this longstanding barrier in the actin cytoskeleton field. Indeed, this novel double-mutant strategy has enabled the PIs' successful determination of the very first atomic crystal structure of actin-nucleator complex (actin-Cobl complex). In addition, a similar approach has been used to solve the structure of actin with a bacterial effector, VopL. The observed non-filament-like conformation in actin-Cobl structure and filament-like conformation in actin-VopL structure together suggest that both types of conformation are fully accessible to an actin complex obtained via the double-mutant strategy, thus its true conformation is most likely preserved. The experiments proposed in this application will apply the double-mutant strategy to three most divergent members of the latest class actin nucleators characterized by the presence of tandem actin-binding sites. The goal is to decipher their molecular mechanisms of actin nucleation, the roles of ATP hydrolysis in their functional cycle, and how they collaborate with specific cellular components to fulfill their functions. This goal will be achieved in three Specific Aims using combined structural and functional approaches: Aim 1: Mechanisms of Cobl-mediated actin nucleation; Aim 2: Mechanistic study of actin nucleation by Lmod; and Aim 3: Mechanistic study of APC-mediated actin nucleation. The extensive preliminary studies presented in this application suggest a high promise of success for the proposed research. Results from this study will be significant not only in elucidating the molecular mechanisms of their respective roles in neurogenesis, muscle development and tumor initiation, but also in unraveling some long-sought-after general underpinnings for de novo actin nucleation, a process underlying every stage of mammalian development as well as many types of pathogenic infection. By providing a detailed atomic gallery of how de novo actin nucleation is accomplished and regulated, this study will stimulate deeper mechanistic investigations on these nucleators as well as discovery and characterization of new actin nucleators. More importantly, this application will validate the double-mutant strategy on proteins/protein fragments with and without actin nucleation activities, thus providing sufficient proof-of-principle for extending this approach to many other actin-involved biological processes beyond actin nucleation. Ultimately, the research along this line is expected to directly benefit the treatment of many forms of human diseases due to actin cytoskeleton malfunctions including neurodegenerative disorders, muscular dystrophy, tumorigenesis and metastasis.